MRI is a technique that allows whole body in vivo imaging in three dimensions at high resolution. In MRI, a static magnetic field is applied to the object of interest while simultaneously or subsequently applying pulses of radio frequency (RF) to change the distribution of the magnetic moments of protons in the object. The change in distribution of the magnetic moments of protons in the object from their equilibrium (normal) distribution to a non-equilibrium distribution and back to the normal distribution (via relaxation processes) constitute the MRI signal.
The longitudinal relaxation time, T1, is defined as the time constant of the exponential recovery of proton spins to their equilibrium distribution along an applied magnetic field after a disturbance (e.g. a RF pulse). The transverse relaxation time, T2, is the time constant that describes the exponential loss of magnetization in a plane transverse to the direction of the applied magnetic field, following a RF pulse that rotates the aligned magnetization into the transverse plane. Magnetic resonance (MR) contrast agents assist this return to a normal distribution by shortening T1 and/or T2 relaxation times.
Signal intensity in biological MRI depends largely on the local value of the longitudinal relaxation rate (1/T1), and the transverse relaxation rate (1/T2) of water protons. Contrast agents will increase 1/T1 and/or 1/T2, depending on the nature of the agent and the strength of the applied field. MRI pulse sequences that emphasize changes in 1/T1 are referred to as T1-weighted and those that emphasize changes in 1/T2 are referred to as T2-weighted. MR contrast agents that include gadolinium (III) ions increase both 1/T1 and 1/T2, and are primarily used with T1-weighted imaging sequences, since the relative change in 1/T1 in tissue is typically much greater than the change in 1/T2. Iron particles, by contrast, provide larger relative changes in 1/T2, and are best visualized in a T2-weighted image.
Advances in MRI have tended to favor T1 agents such as gadolinium based contrast agents. Faster scans with higher resolution require more rapid RF pulsing, and can lead to loss of the MRI signal through saturation effects. T1 agents relieve this saturation and restore signal intensity by stimulating relaxation of nuclear spins between RF pulses.
Because many paramagnetic metal ions, including gadolinium (III), are toxic, they are often administered in a sequestered form, for example, as metal chelates. However, metal chelates, because of their small size and relative hydrophillicity, tend to be cleared rapidly from blood, giving rise to limited cell penetration (but good tolerability). Conjugation of metal chelates to macromolecules to form macromolecular imaging agents is one approach to altering the pharmacological properties (e.g., blood retention, tissue perfusion, and excretion) and biophysical properties (e.g., relaxivity, which is defined as the increase in longitudinal or transverse relaxation rate per millimolar concentration of a contrast agent) of metal chelates. For example, high molecular weight macromolecular imaging agents tend to be retained in the vascular space by virtue of their size, and are useful for blood pool imaging in a technique called magnetic resonance angiography (MRA). Tissue specific accumulation and/or image enhancement are features that may be exhibited by macromolecular contrast agents due to their pharmacokinetic properties, but the mechanisms that lead to such by which this occurs are poorly understood outside of immunologically active contrast agents, such as antibodies conjugated to metal chelates.
Dendrimers are a class of highly branched, often spherical, macromolecular polymers that exhibit greater monodispersity (i.e. a smaller range of molecular weights, sizes, and shapes) than linear polymers of similar size. These three-dimensional oligomeric structures are prepared by reiterative reaction sequences starting from a core molecule that has multiple reactive groups. When monomer units, also having multiple reactive groups, are reacted with the core, the number of reactive groups comprising the outer bounds of the dendrimer increases. Successive layers of monomer molecules may be added to the surface of the dendrimer, with the number of branches and reactive groups on the surface increasing geometrically each time a layer is added. The number of layers of monomer molecules in a dendrimer may be referred to as the “generation” of the dendrimer. The total number of reactive functional groups on a dendrimer's outer surface ultimately depends on the number of reactive groups possessed by the core, the number of reactive groups possessed by the monomers that are used to grow the dendrimer, and the generation of the dendrimer.
The reactive functional groups that form the outer surface of a dendrimer may be conjugated to metal chelates, such as gadolinium (III) chelates, to provide macromolecular MRI contrast agents. Conjugation of multiple metal chelates to a dendrimer core to provide a dendrimer conjugate may provide a contrast agent exhibiting high relaxivities and altered pharmacokinetics relative to the metal chelates themselves. Unfortunately, selection of a dendrimer-based contrast agent that is suitable for imaging a particular tissue or tissue function (by virtue of a combination of distribution to the tissue and image enhancement of particular features of the tissue) is complicated by the current understanding of how molecules are processed and ultimately excreted (e.g. through the kidney and liver) from the body.
In particular, selection of dendrimeric agents for imaging of renal tissue is difficult in view of the complex way in which molecules are processed by renal tissue. For example, blood clearance and renal excretion of low-molecular weight molecules depends on glomerular filtration, which in turn relies on molecular shape and size (See, for example, Chang et al., “Permselectivity of the glomerular capillary wall to macromolecules,” Biophys. J, 15: 887-906 (1975)). Molecular charge also affects renal filtration (See, for example, Guasch et al., “Charge selectivity of the glomerular filtration barrier in healthy and nephrotic humans,” J. Clin. Invest., 92: 2274-2282 (1993)). Once filtered, low-molecular weight molecules generally undergo endocytosis, another process that depends at least in part upon charge (See, for example, Kobayashi et al., “The pharmacokinetic characteristics of glycolated humanized anti-Tac Fabs are determined by their isoelectric points,” Cancer Res., 59: 422-430 (1999)). Furthermore, the relative hydrophobicity or hydrophillicity of a molecule has been shown to affect accumulation in the kidney. For example, a more hydrophobic DAB-Am-64-(1B4M)64 dendrimer contrast agent has been shown to accumulate significantly more in the liver and less in the kidney than a relatively hydrophillic PAMAM-G4D-(1B4M)64 contrast agent (Kobayashi et al., “Novel Liver Macromolecular MR Contrast Agent with a Polypropylenimine Diaminobutyl Dendrimer Core: Comparison to the Vascular MR Contrast Agent with the Polyamidoamine Dendrimer Core,” Magn. Res. Med., 46: 795-802 (2001)). The complicated dependence of renal processing on size, shape, charge and hydrophobicity (hydrophillicity) of a macromolecule makes it difficult to predict which agents will provide contrast for renal structure and function.
Further complicating the discovery of clinically suitable macromolecular MRI contrast agents is the danger that such agents will be retained for extended periods of time in the body, leading to increased potential toxicity from unstable Gd (III) chelates. For example, only 20 percent of the injected dose (% ID) of the PAMAM-G4D based contrast agent was excreted from mice during the first two days following administration (See, Sato et al., “Pharmacokinetics and enhancement patterns of macromolecular MR contrast agents with various sizes of polyamidoamine dendrimer cores,” Magn. Reson. Med. 46: 1169-1173 (2001)). Efforts to increase the excretion rate of dendrimeric contrast agents can lead to altered image enhancement patterns. (See, for example, Kobayashi et. al., “Novel Intravascular Macromolecular MRI Contrast Agent With Generation-4 Polyamidoamine Dendrimer Core: Accelerated Renal Excretion with Coinjection of Lysine,” Magn. Res. Med., 46:457-464 (2001)).